Administrative Supplement ? Abstract Establishing and maintaining apical-basal polarity is essential for epithelial tissue integrity and function under both normal and stressed conditions such as hypoxia/ischemia. A group of so-called polarity proteins play essential and conserved functions in regulating cell polarity in both invertebrates and vertebrates. One key feature of all polarity proteins is that association with plasma membrane (PM) or cell cortex is critical for their in vivo functions. With few exceptions, most polarity proteins are assumed to be localized to PM/cell cortex through protein-protein interactions. Regulatable molecular mechanisms underlying the potential direct interaction between PM and majority of polarity proteins have long been elusive. Polybasic domains that are rich in positively charged Arg and Lyn residues have a well-established role in PM-specific targeting, based on its electrostatic interactions with negatively charged polyphosphoinositides PI4P and PIP2 that are uniquely enriched on the PM inner surface. However, for decades functional studies on polybasic domain in PM targeting have been limited to a small number of proteins and no polybasic domains had been characterized in any of the polarity proteins. We recently identified Lgl as the first polarity protein that contains an evolutionarily conserved and phosphorylatable polybasic domain whose electrostatic binding to PI4P and PIP2 on the PM directly mediate the PM localization of Lgl. In this proposal we have identified that many additional polarity proteins, as well as hundreds to thousands of proteins in Drosophila and human genomes, also contain potential polybasic domains. We will use both Drosophila and cultured mammalian cells to investigate the hypothesis that electrostatic binding between polybasic domain and plasma membrane serves a key regulatable molecular mechanism for controlling the subcellular localizations of multiple polybasic polarity proteins. We will confirm the electrostatic binding of PM by multiple polybasic polarity proteins. More importantly, we will investigate the molecular mechanisms such as phosphorylation, allosteric regulation and coincident protein interactions that may control the direct binding between PM and polybasic proteins to achieve their polarized subcellular localization and activations. Finally, our research highlighted for the first time that hypoxia and depletion of ATP acutely and reversibly inhibit polybasic domain proteins PM targeting through depleting PI4P and PIP2 on the PM. Our research revealed a previously unappreciated but biologically significant challenge for cells to retarget polybasic polarity proteins to original PM domains after hypoxia/ischemia. We will focus on identifying mechanisms that actively direct the retargeting of polybasic polarity proteins to their original PM domains. Our research will establish a new paradigm to understand how regulatable binding between polybasic domains and PM serves as a fundamental mechanism to integrate the protein-protein interactions in achieving polarized PM targeting of polarity protein under both normal and stress conditions.